Surface plasmon-field enhanced fluorescence spectroscopic measurement method and surface plasmon-field enhanced fluorescence spectroscopic measurement device

ABSTRACT

To provide a surface plasmon-field enhanced fluorescence spectroscopic measurement method and a surface plasmon-field enhanced fluorescence spectroscopic measurement device which are capable of accurately measuring a fluorescent signal regardless of the type of a light detection means even when the concentration of an analyte is high by adjusting the dynamic range of the SPFS device. A surface plasmon-field enhanced fluorescence stereoscopic measurement method wherein an analyte labeled with a fluorescent substance is excited by surface plasmon light generated by applying excitation light to a metallic thin film, and generated fluorescence is received by a light detection means to thereby detect the analyte. The dynamic range is expanded by adjusting the amount of the fluorescence received by the light detection means.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. national stage of application No. PCT/JP2012/064089,filed on 31 May 2012. Priority under 35 U.S.C. §119 (a) and 35 U.S.C.§365(b) is claimed from Japanese Application No. 2011-135511, filed 17Jun. 2011, the entirety of which is also incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to a surface plasmon-field enhancedfluorescence spectroscopic measurement method and a surfaceplasmon-field enhanced fluorescence spectroscopic measurement apparatusbased on a principle of a surface plasmon-field enhanced fluorescencespectroscopy (SPFS) for putting a surface plasmon resonance (SPR)phenomenon to practical use.

BACKGROUND ART

In the case in which a detection of an extremely fine substance iscarried out, a wide variety of specimen material detection apparatus hasbeen used for enabling an inspection of such a substance by putting aphysical phenomenon of a substance to practical use from the past.

As one of such specimen material detection apparatuses, there can bementioned for instance a surface plasmon resonance apparatus (hereafterreferred to as an SPR apparatus) in which a phenomenon of a resonance ofan electron and a light in a minute region of a nanometer level or thelike (a surface plasmon resonance (SPR) phenomenon) is put to practicaluse and an extremely fine analyte in a biological body is detected forinstance.

As one of such specimen material detection apparatuses, there also canbe mentioned for instance a surface plasmon-field enhanced fluorescencespectroscopic measurement apparatus (hereafter referred to as an SPFSapparatus) in which the analyte detection can be carried out with ahigher degree of accuracy as compared with the SPR apparatus based on aprinciple of a surface-plasmon enhanced fluorescence spectroscopy (SPFS)for putting a surface plasmon resonance (SPR) phenomenon to practicaluse.

For the surface-plasmon enhanced fluorescence spectroscopy (SPFS), underthe condition of the attenuated total reflectance (ATR) of an excitationlight such as a laser light that has been applied from the light sourceon a surface of a metallic thin film, by generating a surface plasmonlight (a crude density wave) on a surface of a metallic thin film, aphoton amount that is included in an excitation light that has beenapplied from the light source is increased by several ten times toseveral hundred times to obtain an electric field enhancement effect ofa surface plasmon light.

By the electric field enhancement effect, a fluorescence substance thathas been coupled (labeled) with an analyte that has been captured near ametallic thin film is excited in an efficient manner. By observing thefluorescence while using a light detection means such as aphotomultiplier tube (PMT) of a photon counting system and a chargecoupled device (CCD) camera, an analyte of an infinitesimal quantityand/or an extremely low concentration is detected in the above method.

PRIOR ART DOCUMENTS Patent Literature [Patent Literature 1]

-   Japanese Patent Application Laid-Open Publication No. 2009-79970

[Patent Literature 2]

-   Japanese Patent Application Laid-Open Publication No. 2009-244270

SUMMARY OF INVENTION Problems to be Solved by the Invention

However, in the case of an SPFS apparatus that is provided with highsensitivity in which an analyte is integrated in an assay area (ameasurement region), a fluctuation range of a fluorescence signal (asignal that is output from a light detection means) is extremely largeas compared with a change of a concentration of an analyte.

Consequently, in the case in which a concentration of an analyte ishigh, a dynamic range is exceeded for a light detection means such as aphotomultiplier tube and a CCD camera, whereby a measurement cannot becarried out in a precise manner in some cases.

In the case of a camera in which an imaging element such as a CCD isused for instance, as shown in FIG. 14, in the case in which a light ofa certain level or higher is received, a light receiving element issaturated and an actual amount of fluorescence cannot be measuredunfortunately. In addition, in the case in which a photomultiplier tubeof a photon counting system is used, as shown in FIG. 15, in the case inwhich a light of a certain level or higher is received, a count losscaused by a pulse overlap occurs and a fluorescence signal is reducedunfortunately.

The Patent Literature 1 discloses that a dynamic range is tried to beexpanded by changing an area of a measurement region on a sample platein order to restrict a number of substances to be analyzed in ameasurement volume.

The Patent Literature 2 discloses a wide variety of methods forexpanding a dynamic range such as a method in which a dynamic range of aspecimen material analysis is expanded by synthesizing a plurality ofsignals that are provided with different exposure time and a calculationand a processing of a signal for quantifying an integrated light.

However, for the method that is disclosed in the Patent Literature 1, ameasurement region that is provided with a different area is required inaccordance with a concentration of an analyte, whereby an SPFS apparatuswill be growing in size. Or every when an analyte that is provided witha different concentration is measured, it is necessary to prepare asample plate in which a measurement region that is provided with adifferent area is formed.

On the other hand, as a problem peculiar to an SPFS apparatus, in thecase in which a fluorescence labeled substance is linked to an analytethat has been integrated in an assay area, a fluorescence substance isclosely spaced in a limited area, whereby a concentration quenchingoccurs and a fluorescence signal is reduced. Or a fluorescence that hasbeen emitted from a fluorescence substance that has been linked to ananalyte is coupled to a surface plasmon light that has been generated ona surface of a metallic thin film, causing a loss of the fluorescencesignal in some cases.

It is important in order to secure the accuracy of an SPFS apparatus todiscriminate whether the loss of the fluorescence signal occurs on ametallic thin film or a fluorescence signal is reduced by a problem of adynamic range of a light detection means. However, the inventions thathave been disclosed in the Patent Literatures 1 and 2 is not necessarilyrelated to an expansion of a dynamic range that is suitable for an SPFSapparatus in a practical sense.

The present invention was made in consideration of such conditions, andan object of the present invention is to provide a surface plasmon-fieldenhanced fluorescence spectroscopic measurement method and surfaceplasmon-field enhanced fluorescence spectroscopic measurement apparatusthat are capable of measuring a fluorescence signal in a precise mannerregardless of a type of a light detection means by adjusting a dynamicrange of an SPFS apparatus even in the case in which a concentration ofan analyte is high.

Means for Solving the Problems

The present invention was made in order to solve the problems of theconventional art described above and achieve the purpose.

A surface plasmon-field enhanced fluorescence spectroscopic measurementmethod in accordance with the present invention is characterized bycomprising the steps of exciting a fluorescence substance that haslabeled an analyte by surface plasmon light that has been generated byapplying an excitation light to a metallic thin film and receiving thegenerated fluorescence by a light detection means to thereby detect theanalyte, wherein:

a dynamic range is expanded by adjusting a light amount of thefluorescence that is received by the light detection means.

A surface plasmon-field enhanced fluorescence spectroscopic measurementapparatus in accordance with the present invention that is configured tocarry out a detection of a specimen material by applying an excitationlight, in which a metallic thin film that is formed on a dielectricmember, a fine flow passage that is formed on an upper surface of themetallic thin film, and a sensor chip that is provided with a sensorpart that is formed in the fine flow passage are mounted, ischaracterized by comprising:

a light source that is configured to apply an excitation light to themetallic thin film via the dielectric member; and

a light detection means that is disposed over the sensor chip,

wherein the light detection means is configured to receive afluorescence that is generated by exciting a fluorescence substance thatlabels an analyte that is fixed to the sensor part by a surface plasmonlight that is generated in the case in which the excitation light isapplied to the metallic thin film; and

a fluorescence amount adjusting means is configured to be able to adjusta light amount of a fluorescence that is received by the light detectionmeans.

By this configuration, in the case in which a detection of a specimenmaterial solution that is provided with a high concentration of ananalyte is carried out, even in the case in which a fluorescence that isprovided with a light amount that cannot be measured by the lightdetection means is emitted from a fluorescence substance, a measurementcan be carried out in a precise manner by adjusting an amount of afluorescence that is received by the light detection means, and adetection of an analyte that is provided with a wide dynamic range canbe carried out in a precise manner.

In addition, for the present invention, since a light amount of afluorescence that is received by the light detection means is adjusted,a detection of an analyte that is provided with a wide dynamic range canbe carried out in a precise manner regardless of a type of a lightdetection means.

The surface plasmon-field enhanced fluorescence stereoscopic measurementmethod in accordance with the present invention is characterized byfurther comprising the steps of:

by comparing a first fluorescence signal that is output in the case inwhich the light detection means receives a fluorescence under a firstcondition and a second fluorescence signal that is output in the case inwhich the light detection means receives a fluorescence in which a lightamount has been adjusted under a second condition in which a lightamount of a fluorescence that is received by the light detection meansis adjusted so as to be smaller than that in the first condition,judging whether or not the first fluorescence signal is abnormal; and

obtaining a normal fluorescence signal by correcting the secondfluorescence signal in the case in which it is decided that the firstfluorescence signal is abnormal.

The surface plasmon-field enhanced fluorescence spectroscopicmeasurement apparatus in accordance with the present invention ischaracterized in that:

by comparing a first fluorescence signal that is output in the case inwhich the light detection means receives a fluorescence under a firstcondition and a second fluorescence signal that is output in the case inwhich the light detection means receives a fluorescence in which a lightamount has been adjusted by the fluorescence amount adjusting meansunder a second condition in which a light amount of a fluorescence thatis received by the light detection means is adjusted so as to be smallerthan that in the first condition, it is judged whether or not the firstfluorescence signal is abnormal; and

a normal fluorescence signal is obtained by correcting the secondfluorescence signal in the case in which it is decided that the firstfluorescence signal is abnormal.

By this configuration, even in the case in which a fluorescence that isprovided with a light amount that cannot be measured by the lightdetection means is emitted from a fluorescence substance, a normalfluorescence signal can be obtained by using a second fluorescencesignal that is output in the case in which the light detection meansreceives a fluorescence in which a light amount has been adjusted.

Consequently, a detection can be carried out in a precise manner forspecimen material solutions including a specimen material solution thatis provided with a high concentration of an analyte and a specimenmaterial solution that is provided with a low concentration of ananalyte, and a detection of an analyte that is provided with a widedynamic range can be carried out.

The surface plasmon-field enhanced fluorescence stereoscopic measurementmethod in accordance with the present invention is characterized byfurther comprising the step of adjusting a light amount of afluorescence that is received by the light detection means by adjustinga light amount of the fluorescence that has been generated.

The surface plasmon-field enhanced fluorescence spectroscopicmeasurement apparatus in accordance with the present invention ischaracterized in that the fluorescence amount adjusting means isdisposed between the sensor chip and the light detection means.

By this configuration, a fluorescence that is emitted by exciting afluorescence substance by a surface plasmon light can be adjusted in adirect way, and it is easy to reduce a light amount to be a light amountthat can be measured by the light detection means.

The surface plasmon-field enhanced fluorescence stereoscopic measurementmethod in accordance with the present invention is characterized byfurther comprising the step of adjusting a light amount of afluorescence that is received by the light detection means by adjustinga light amount of the excitation light.

The surface plasmon-field enhanced fluorescence spectroscopicmeasurement apparatus in accordance with the present invention ischaracterized in that the fluorescence amount adjusting means isdisposed between the light source and the dielectric member.

Even in the case of this configuration, the intensity of an electricalfield by a surface plasmon light can be reduced by reducing a lightamount of the excitation light that is applied to the metallic thinfilm, and as a result, a light amount of a fluorescence that is emittedfrom a fluorescence substance that labels an analyte can be reduced tobe a light amount that can be measured by the light detection means.

The surface plasmon-field enhanced fluorescence stereoscopic measurementmethod in accordance with the present invention is characterized byfurther comprising the step of:

receiving a fluorescence by the light detection means while changing anincidence angle of the excitation light to the metallic thin film in apredetermined range; and

judging an abnormal fluorescence signal based on the relationshipbetween an incidence angle of the excitation light and a fluorescencesignal that is output in the case in which the light detection meansreceives a fluorescence.

The surface plasmon-field enhanced fluorescence spectroscopicmeasurement apparatus in accordance with the present invention ischaracterized in that:

a fluorescence is received by the light detection means while changingan incidence angle of the excitation light to the metallic thin film ina predetermined range by using the application angle adjusting means;and

an abnormal fluorescence signal is judged based on the relationshipbetween an incidence angle of the excitation light and a fluorescencesignal that is output in the case in which the light detection meansreceives a fluorescence.

Even in the case of this configuration, in the case in which afluorescence that is provided with a light amount that cannot bemeasured by the light detection means is emitted from a fluorescencesubstance, it can be decided that an abnormal fluorescence signal isoutput from the light detection means.

Advantageous Effects of Invention

By the present invention, a measurement can be carried out in a precisemanner and an analyte that is provided with a wide dynamic range that issuitable for a surface plasmon-field enhanced fluorescence spectroscopicmeasurement method or a surface plasmon-field enhanced fluorescencespectroscopic measurement apparatus by adjusting an amount of afluorescence that is received by a light detection means by using afluorescence amount adjusting means even in the case in which afluorescence that exceeds a measurement enable range of a lightdetection means is generated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic plan view showing a frame format of a simpleoverview of a surface plasmon-field enhanced fluorescence spectroscopicmeasurement apparatus for describing a surface plasmon-field enhancedfluorescence spectroscopic measurement method in accordance with anembodiment of the present invention.

FIG. 2 is a partially enlarged view of FIG. 1.

FIG. 3 is a flowchart for illustrating a flow of a surface plasmon-fieldenhanced fluorescence spectroscopic measurement.

FIG. 4 is a graph for showing the relationship between a concentrationof an analyte and a fluorescence signal that is output from a lightdetection means 30 in a state in which a light intensity is not reducedand a fluorescence signal that is output from a light detection means 30in a state in which a light intensity is reduced.

FIG. 5 is a graph for showing the relationship between a concentrationof an analyte and a fluorescence signal that is output from a lightdetection means 30 in a state in which a correction is not carried outand a fluorescence signal that is output from a light detection means 30in a state in which a correction is carried out.

FIG. 6 is a schematic plan view showing a frame format of a simpleoverview of an SPFS apparatus for describing a surface plasmon-fieldenhanced fluorescence spectroscopic measurement method in accordancewith an embodiment of the present invention.

FIG. 7 is a graph for showing the relationship between a concentrationof an analyte and a fluorescence signal that is output from a lightdetection means 30 in a state in which a light intensity is not reducedand a fluorescence signal that is output from a light detection means 30in a state in which a light intensity is reduced.

FIG. 8 is a graph for showing the relationship between a concentrationof an analyte and a fluorescence signal that is output from a lightdetection means 30 in a state in which a correction is not carried outand a fluorescence signal that is output from a light detection means 30in a state in which a correction is carried out.

FIG. 9 is a schematic plan view showing a frame format of a simpleoverview of an SPFS apparatus for describing a surface plasmon-fieldenhanced fluorescence spectroscopic measurement method in accordancewith an embodiment of the present invention.

FIG. 10 is a schematic plan view showing a frame format of a simpleoverview of an SPFS apparatus for describing a surface plasmon-fieldenhanced fluorescence spectroscopic measurement method in accordancewith an embodiment of the present invention.

FIG. 11 is a partially enlarged view of FIG. 10.

FIG. 12 is a graph for showing the relationship between an incidenceangle of an excitation light and a fluorescence signal in the case inwhich a specimen material solution that is provided with a lowconcentration of an analyte is measured.

FIG. 13 is a graph for showing the relationship between an incidenceangle of an excitation light and a fluorescence signal in the case inwhich a specimen material solution that is provided with a highconcentration of an analyte is measured.

FIG. 14 is a graph for showing the relationship between a light amountof a received fluorescence and a fluorescence signal in the case inwhich a CCD camera is used.

FIG. 15 is a graph for showing the relationship between a light amountof a received fluorescence and a fluorescence signal in the case inwhich a photomultiplier tube of a photon counting system is used.

DESCRIPTION OF EMBODIMENTS

An embodiment (example) of the present invention will be described belowin more detail with reference to the drawings.

Embodiment 1 1. Embodiment in the Case in which a Light Amount of aFluorescence that has been Generated is Adjusted

FIG. 1 is a schematic plan view showing a frame format of a simpleoverview of a surface plasmon-field enhanced fluorescence spectroscopicmeasurement apparatus (hereafter referred to as an SPFS apparatus) fordescribing a surface plasmon-field enhanced fluorescence spectroscopicmeasurement method in accordance with an embodiment of the presentinvention. FIG. 2 is a partially enlarged view of FIG. 1. FIG. 3 is aflowchart for illustrating a flow of a surface plasmon-field enhancedfluorescence spectroscopic measurement.

1-1. Configuration of the SPFS Apparatus

An SPFS apparatus 10 in accordance with the present invention isprovided with a sensor chip 18 that is composed of a dielectric member12 in a prism shape in which a vertical cross sectional shape is agenerally trapezoidal shape, a metallic thin film 14 that is formed on ahorizontal upper surface 12 a of the dielectric member 12, and a fineflow passage 16 that is formed on a horizontal upper surface 14 a of themetallic thin film 14. The sensor chip 18 is mounted to a sensor chipmounting part 20 of the SPFS apparatus 10.

Moreover, a sensor part 22 to which a ligand that is linked to aparticular analyte in a specific manner has been fixed is formed at apart of the fine flow passage 16. A specimen material solution thatincludes a particular analyte is made inflow into the sensor part 22 viathe fine flow passage 16, and a fluorescence substance that labels ananalyte is then made inflow via the fine flow passage 16. By thisconfiguration, an analyte that has been labeled by a fluorescencesubstance can be fixed to the sensor part 22.

A fluorescence substance is not restricted in particular as long as afluorescence substance is a substance that is excited and that emits thefluorescence in the case in which a predetermined excitation light isapplied or an electrical field effect is utilized. In the presentspecification, the “fluorescence” includes a wide variety of emissionsof lights such as phosphorescence.

A specimen material solution that includes an analyte is not restrictedin particular. As such a specimen material, there can be mentioned forinstance a blood, a blood serum, a blood plasma, urine, a nasal passagefluid, a saliva, a feces, and a body cavity fluid (such as a spinalfluid, an ascites fluid, and a pleural effusion).

As an analyte that is included in a specimen material, there can bementioned for instance a nucleic acid (single-stranded ordouble-stranded DNA,RNA, polynucleotide, oligonucleotide, and PNA(peptide nucleic acid), and nucleoside, nucleotide, and a modifiedmolecule thereof), a protein substance (such as polypeptide andoligopeptide), an amino acid (including a modified amino acid),carbohydrate (such as oligosaccharide, polysaccharide, and a sugarchain), lipid, a modified molecule thereof, and a complex thereof. Morespecifically, an analyte can also be a carcinoembryonic antigen such asan AFP (α fetoprotein), a tumor marker, a signal transducer, and ahormone, and is not restricted in particular.

A light source 24 is disposed on a side of one side face 12 b under thedielectric member 12 as shown in FIG. 1. An excitation light 26 from thelight source 24 is incident to the side face 12 b of the dielectricmember 12 from the outside and the lower side of the dielectric member12. The excitation light 26 is then applied toward the metallic thinfilm 14 that has been formed on an upper surface 12 a of the dielectricmember 12 at a predetermined incidence angle (a resonance angle) α1 bywhich the attenuated total reflectance (ATR) of the excitation light 26occurs via the dielectric member 12.

A light detection means 30 that is configured to receive a fluorescence28 that is emitted by an excitation of a fluorescence substance isdisposed over the sensor chip 18.

The light detection means 30 is not restricted in particular. As thelight detection means 30, a photomultiplier tube of a photon countingsystem, a CCD (Charge Coupled Device) image sensor capable of performinga multipoint measurement, and a CMOS (Complementary Metal OxideSemiconductor) image sensor can be used for instance. In the presentembodiment, a photomultiplier tube of a photon counting system is used.

In the present embodiment, as a fluorescence amount adjusting means 32that is configured to adjust a light amount of a fluorescence that hasemitted, a neutral density (ND) filter 34 that is configured to be ableto cut a light amount of 99% is disposed between the sensor chip 18 andthe light detection means 30 in such a manner that the filter can bemoved into or out without any inhibition.

The fluorescence amount adjusting means 32 is not restricted inparticular as long as the fluorescence amount adjusting means 32 canadjust an amount of a light that is received for the light detectionmeans 30. For far more than the neutral density (ND) filter 34, awavelength selective filter or a diaphragm lens can also be disposed insuch a manner that the wavelength selective filter or the diaphragm lenscan be moved into or out without any inhibition. In addition, anaperture can also be formed and a light amount can be adjusted dependingon a size of an opening of the aperture.

In the present embodiment, the fluorescence amount adjusting means 32can also be made to be a focus adjusting means of the light detectionmeans 30. By defocusing the light detection means 30, an amount of afluorescence that is received by the light detection means 30 can alsobe adjusted.

In the present embodiment, an excitation light is applied from the lightsource 24 is not restricted in particular. However, it is preferable touse an excitation light of a wavelength in the range of 200 to 900 nmand of the range of 0.001 to 1000 mW, more preferably, an excitationlight of a wavelength in the range of 230 to 800 nm and of the range of0.01 to 100 mW.

The dielectric member 12 is not restricted in particular. As thedielectric member 12, a wide variety of inorganic substances such as aglass and a ceramics, natural polymers, and synthetic polymers that areoptically transparent can be used. From the aspect of the chemicalstability, manufacturing stability, and optical transparency, it ispreferable that the dielectric member 12 includes silicon dioxide (SiO₂)or titanium dioxide (TiO₂).

The dielectric member 12 in a prism shape in which a vertical crosssectional shape is a generally trapezoidal shape is used in the presentembodiment. However, a shape of the dielectric member 12 can also bemodified in an appropriate manner in such a manner that a vertical crosssectional shape is a triangular shape (so-called a triangular prism), asemicircular shape, and a semi elliptical shape.

A material of the metallic thin film 14 is not restricted in particular.As a material of the metallic thin film 14, the metallic thin film 14 ismade of a metal of at least one kind that is selected from a group thatis composed of gold, silver, aluminum, copper, and platinum, morepreferably gold, and the metallic thin film 14 can also be made of analloy of the metal.

Such a metal is stable to oxidization and is suitable for the metallicthin film 14 since an electrical field enhancement caused by a surfaceplasmon light (a crude density wave) is increased as described later.

A method for forming the metallic thin film 14 is not restricted inparticular. As a method for forming the metallic thin film 14, there canbe mentioned for instance a sputtering method, a vapor deposition method(such as a resistance heating vapor deposition method and an electronbeam vapor deposition method), an electrolytic plating method, and anelectroless plating method. In particular, a sputtering method and avapor deposition method are preferable since an adjustment of thecondition of a thin film formation can be easily carried out.

A thickness of the metallic thin film 14 is not restricted inparticular. As a thickness of the metallic thin film 14, it ispreferable that a thickness of gold is in the range of 5 to 500 nm, athickness of silver is in the range of 5 to 500 nm, a thickness ofaluminum is in the range of 5 to 500 nm, a thickness of copper is in therange of 5 to 500 nm, a thickness of platinum is in the range of 5 to500 nm, and a thickness of an alloy of the metal is in the range of 5 to500 nm.

From the aspect of an electrical field enhancement effect as describedlater, as a more preferable thickness of the metallic thin film 14, itis more preferable that a thickness of gold is in the range of 20 to 70nm, a thickness of silver is in the range of 20 to 70 nm, a thickness ofaluminum is in the range of 10 to 50 nm, a thickness of copper is in therange of 20 to 70 nm, a thickness of platinum is in the range of 20 to70 nm, and a thickness of an alloy of the metal is in the range of 20 to70 nm.

In the case in which a thickness of the metallic thin film 14 is in therange described above, the thickness is suitable since a surface plasmonlight (a crude density waven) described later is easily generated. Forthe metallic thin film 14 that is provided with such a thickness, thedimensions and a shape of a size (vertical length×horizontal length) arenot restricted in particular.

1-2. Method for Measuring a Fluorescence Amount

A surface plasmon-field enhanced fluorescence spectroscopic measurementmethod using the SPFS apparatus 10 in accordance with the presentinvention that is configured as described above will be described inaccordance with the flowchart shown in FIG. 3.

In the first place, a specimen material solution that includes aparticular analyte is made inflow into the sensor part 22 via the fineflow passage 16, and a fluorescence substance that labels the analyte isthen made inflow via the fine flow passage 16 similarly. By thisconfiguration, an analyte that has been labeled by a fluorescencesubstance is fixed to the sensor part 22.

An excitation light 26 is applied from the light source 24 in thisstate, and the excitation light 26 is incident to the side face 12 b ofthe dielectric member 12 from the outside and the lower side of thedielectric member 12. The excitation light 26 is then applied toward themetallic thin film 14 that has been formed on an upper surface 12 a ofthe dielectric member 12 at a predetermined incidence angle (a resonanceangle) α1 by which the attenuated total reflectance (ATR) of theexcitation light 26 occurs via the dielectric member 12.

By applying the excitation light 26, a surface plasmon light (a crudedensity waven) is emitted from the surface of the metallic thin film 14,and a fluorescence substance that labels the analyte, which has beenfixed to the sensor part 22, is excited by the surface plasmon light (acrude density waven), thereby emitting a fluorescence 28.

At this time, the fluorescence amount adjusting means 32 is in the statein which a light intensity is not reduced, that is, the neutral density(ND) filter 34 is not inserted between the sensor chip 18 and the lightdetection means 30 (a first condition).

By detecting the fluorescence 28 by using the light detection means 30,the analyte that has been fixed to the sensor part 22 is detected and afluorescence signal in accordance with a concentration of an analyte (afirst fluorescence signal) is output from the light detection means 30.

In the next place, in the state in which a light intensity is reduced bythe fluorescence amount adjusting means 32, that is, the neutral density(ND) filter 34 is inserted between the sensor chip 18 and the lightdetection means 30 (a second condition), the fluorescence 28 is detectedby using the light detection means 30. As a result, a secondfluorescence signal is output from the light detection means 30.

By reducing a light by the fluorescence amount adjusting means 32, thefluorescence 28 that is received by the light detection means 30 becomes1/100 as compared with the state in which a light intensity is notreduced. Consequently, in the case in which the light detection means 30receives the fluorescence 28 in a normal way, the second fluorescencesignal also becomes 1/100 as compared with the first fluorescence signalthat is output in the state in which a light intensity is not reduced.

Based on this, in the case in which the second fluorescence signal inthe state in which a light intensity is reduced is larger than 1/100 ofthe first fluorescence signal in the state in which a light intensity isnot reduced, an accurate fluorescence signal can be obtained bycorrecting the second fluorescence signal in the state in which a lightintensity is reduced as described later.

1-3. Correction of a Fluorescence Signal

Table 1 shows the relationship between a concentration of an analyte anda fluorescence signal that is output by the light detection means 30 inthe state in which a light intensity is not reduced and a fluorescencesignal that is output by the light detection means 30 in the state inwhich a light intensity is reduced. FIG. 4 is a graph indicating therelationship of Table 1.

TABLE 1 Concentration of an analyte [g/mL] Fluorescence signal[counts/sec] Light intensity is not reduced Light intensity is reduced

As shown in Table 1 and FIG. 4, in the case in which a light intensityis not reduced by the fluorescence amount adjusting means 32, the highera concentration of an analyte is, the lower a level of a fluorescencesignal is by contraries.

The judgment of a reduction of a fluorescence signal is carried outbased on that a fluorescence signal in the case in which a lightintensity is reduced by the fluorescence amount adjusting means 32becomes 1/100 as compared with a fluorescence signal in which a lightintensity is not reduced as described above.

In other words, in the present embodiment, in the case in which aconcentration of an analyte is high, that is, in the range of 1.0E-9 to1.0E-7 (g/mL), a fluorescence signal in the case in which a lightintensity is reduced is larger than 1/100 of a fluorescence signal inthe case in which a light intensity is not reduced.

In this case, an accurate value of a fluorescence signal can be obtainedby correcting a value while using a fluorescence signal in the case inwhich a light intensity is reduced (in the present embodiment, a valueof a fluorescence signal in the case in which a light intensity isreduced is centupled).

On the other hand, in the case in which a concentration of an analyte islow, that is, in the range of 1.0E-13 to 1.0E-12 (g/mL), a fluorescencesignal in the case in which a light intensity is reduced is also largerthan 1/100 of a fluorescence signal in the case in which a lightintensity is not reduced. This is caused by the lowest value of a lightamount that can be measured by the light detection means 30, and this isbecause a fluorescence signal in the case in which a light intensity isreduced outputs an abnormal value.

Consequently, in the case in which a concentration of an analyte is low,a fluorescence signal in the case in which a light intensity is notreduced can be obtained as a normal value.

As described above, a normal measurement of a concentration of ananalyte can be carried out by using a corrected fluorescence signal asshown in Table 2 and FIG. 5.

TABLE 2 Concentration of an analyte [g/mL] Fluorescence signal[counts/sec] Correction is not carried out Correction is carried out

Embodiment 2 2. Embodiment in the Case in which an Amount of anExcitation Light is Adjusted

FIG. 6 is a schematic plan view showing a frame format of a simpleoverview of an SPFS apparatus for describing a surface plasmon-fieldenhanced fluorescence spectroscopic measurement method in accordancewith an embodiment of the present invention.

The SPFS apparatus 10 of the modified example is provided with aconfiguration that is equivalent to that of the SPFS apparatus shown inFIGS. 1 to 5 in a basic way and the principle of the SPFS apparatus 10is also equivalent to that of the SPFS apparatus shown in FIGS. 1 to 5in a basic way. Consequently, constitutive members that are equivalentto those illustrated in FIGS. 1 to 5 are numerically numbered similarlyand the detailed descriptions of the equivalent constitutive members areomitted.

2-1. Configuration of the SPFS Apparatus

As shown in FIG. 6, the modified example is provided with a fluorescenceamount adjusting means 32 between a light source 24 and a sensor chip18. By this configuration, a light amount of an excitation light 26 thatis applied from the light source 24 to the metallic thin film 14 can beadjusted (reduced), and as a result, the intensity of an electricalfield by a surface plasmon light that is generated on the surface of themetallic thin film 14 can be reduced.

Consequently, a light amount of a fluorescence that is emitted from afluorescence substance that labels an analyte can be adjusted, andsimilarly to the embodiment 1, a light amount of a fluorescence that isreceived by the light detection means 30 can be adjusted. Moreover, afluorescence signal in accordance with a concentration of an analyte canbe obtained by using a measurement method of a light amount of thefluorescence similar to the embodiment 1

In the case of the present embodiment, the fluorescence amount adjustingmeans 32 can also be made to be a light amount adjusting function of thelight source 24, and a light amount of a fluorescence 26 that is emittedfrom the light source 24 can also be adjusted.

2-2. Correction of a Fluorescence Signal

Table 3 shows the relationship between a concentration of an analyte anda fluorescence signal that is output by the light detection means 30 inthe state in which a light intensity is not reduced and a fluorescencesignal that is output by the light detection means 30 in the state inwhich a light intensity is reduced. FIG. 7 is a graph indicating therelationship of Table 3.

TABLE 3 Concentration of an analyte [g/mL] Fluorescence signal[counts/sec] Light intensity is not reduced Light intensity is reduced

As shown in Table 3 and FIG. 7, in the case in which an excitation lightis not reduced by the fluorescence amount adjusting means 32, as aconcentration of an analyte is higher, a plateaued abnormal output isgenerated.

The judgment of an abnormal output of a fluorescence signal is carriedout based on that a fluorescence signal in the case in which a lightintensity is reduced by the fluorescence amount adjusting means 32becomes 1/100 as compared with a fluorescence signal in which a lightintensity is not reduced similarly to the embodiment 1.

In other words, in the present embodiment, in the case in which aconcentration of an analyte is high, that is, in the range of 1.0E-9 to1.0E-7 (g/mL), a fluorescence signal in the case in which a lightintensity is reduced is larger than 1/100 of a fluorescence signal inthe case in which a light intensity is not reduced.

In this case, an accurate value of a fluorescence signal can be obtainedby correcting a value while using a fluorescence signal in the case inwhich a light intensity is reduced (in the present embodiment, a valueof a fluorescence signal in the case in which a light intensity isreduced is centupled).

On the other hand, in the case in which a concentration of an analyte islow, that is, in the range of 1.0E-13 to 1.0E-12 (g/mL), a fluorescencesignal in the case in which a light intensity is reduced is also largerthan 1/100 of a fluorescence signal in the case in which a lightintensity is not reduced. This is caused by the lowest value of a lightamount that can be measured by the light detection means 30, and this isbecause a fluorescence signal in the case in which a light intensity isreduced outputs an abnormal value.

Consequently, in the case in which a concentration of an analyte is low,a fluorescence signal in the case in which a light intensity is notreduced can be obtained as a normal value.

As described above, a normal measurement of a concentration of ananalyte can be carried out by using a corrected fluorescence signal asshown in Table 4 and FIG. 8.

TABLE 4 Concentration of an analyte [g/mL] Fluorescence signal[counts/sec] Correction is not carried out Correction is carried out

Embodiment 3 3. Embodiment in the Case in which a Light Amount of aFluorescence that has been Generated and an Amount of an ExcitationLight are adjusted

FIG. 9 is a schematic plan view showing a frame format of a simpleoverview of an SPFS apparatus for describing a surface plasmon-fieldenhanced fluorescence spectroscopic measurement method in accordancewith an embodiment of the present invention.

The SPFS apparatus 10 of the modified example is provided with aconfiguration that is equivalent to that of the SPFS apparatus shown inFIGS. 1 to 5 in a basic way and the principle of the SPFS apparatus 10is also equivalent to that of the SPFS apparatus shown in FIGS. 1 to 5in a basic way. Consequently, constitutive members that are equivalentto those illustrated in FIGS. 1 to 5 are numerically numbered similarlyand the detailed descriptions of the equivalent constitutive members areomitted.

3-1. Configuration of the SPFS Apparatus

As shown in FIG. 9, the modified example is provided with a fluorescenceamount adjusting means (a fluorescence amount adjusting means 32 a)between a sensor chip 18 and a light detection means 30, and afluorescence amount adjusting means (a fluorescence amount adjustingmeans 32 b) between a light source 24 and a sensor chip 18.

By this configuration, a light amount of a fluorescence that is emittedfrom a fluorescence substance that labels an analyte can be adjusted bythe fluorescence amount adjusting means 32 a, and a light amount of anexcitation light 26 that is applied from the light source 24 to themetallic thin film 14 can be adjusted.

In addition to an adjustment of a light amount of a fluorescence that isgenerated, by adjusting a light amount of an excitation light 26 that iscaused by a generation of a fluorescence 28, a wider dynamic range canbe provided.

For the SPFS apparatus 10 in accordance with the present invention thatis configured as described above, similarly to the embodiments 1 and 2,by detecting an analyte that has been fixed to the sensor part 22 whileadjusting a light amount of a fluorescence, a fluorescence signal in awider dynamic range can be obtained, and a measurement of a specimenmaterial solution that is provided with a concentration of an analyte ina wider range is carried out.

Embodiment 4 4. Embodiment in the Case in which a Light Source isProvided with an Application Angle Adjusting Means

FIG. 10 is a schematic plan view showing a frame format of a simpleoverview of an SPFS apparatus for describing a surface plasmon-fieldenhanced fluorescence spectroscopic measurement method in accordancewith an embodiment of the present invention.

The SPFS apparatus 10 of the modified example is provided with aconfiguration that is equivalent to that of the SPFS apparatus shown inFIGS. 1 to 5 in a basic way and the principle of the SPFS apparatus 10is also equivalent to that of the SPFS apparatus shown in FIGS. 1 to 5in a basic way. Consequently, constitutive members that are equivalentto those illustrated in FIGS. 1 to 5 are numerically numbered similarlyand the detailed descriptions of the equivalent constitutive members areomitted.

4-1. Configuration of the SPFS Apparatus

As shown in FIG. 10, the modified example is provided with anapplication angle adjusting means that is configured to be able tochange an incidence angle of the excitation light 26 that is appliedtoward the metallic thin film 14 to the metallic thin film 14 via thedielectric member 12 from the light source 24.

In general, it is known that an angle dependency as shown in FIG. 12exists for an incidence angle to the metallic thin film 14 and afluorescence signal.

In the present embodiment, a light amount of the fluorescence can bereduced by making an incidence angle of the excitation light 26 to themetallic thin film 14 smaller than a resonance angle (that is, an angleby which the attenuated total reflectance (ATR) of the excitation light26 occurs) or making the incidence angle larger than the resonance anglebased on the angle dependency.

4-2. Correction of a Fluorescence Signal

In the case in which the light source 24 is provided with an applicationangle adjusting means like the modified example, an incidence angle asdescribed above can also be used for judging an abnormal output of afluorescence signal.

In other words, in the case in which a detection of an analyte iscarried out, by obtaining a fluorescence signal while changing anincidence angle of the excitation light 26 in a predetermined range (inthe present embodiment, in a range of 45° to 70°), a relationshipbetween an incidence angle of the excitation light and a fluorescencesignal can be obtained as shown in FIG. 12.

Under normal conditions, as a concentration of an analyte becomeshigher, a fluorescence signal is increased. However, in the case inwhich a concentration of an analyte becomes high and a light amount of afluorescence that is emitted exceeds a dynamic range of the lightdetection means 30, an abnormal fluorescence signal is obtained in sucha manner that a plurality of peaks are generated as shown in FIG. 13.

In the case in which an abnormal fluorescence signal is obtained,similarly to the embodiments 1 to 3, a detection of an analyte that isprovided with a wide dynamic range can be carried out in a precisemanner by correcting a fluorescence signal that has been obtained in thecase in which a light amount of a fluorescence that is received by thelight detection means 30 is reduced by using the fluorescence amountadjusting means 32.

While the preferred embodiments in accordance with the present inventionhave been described above, the present invention is not restricted tothe embodiments described above. In the above described embodiment forinstance, a light intensity is reduced by the fluorescence amountadjusting means 32 in such a manner that a light amount of thefluorescence 28 that is received by the light detection means 30 becomes1/100. However, some changes can be carried out in an appropriate mannerin accordance with a dynamic range that is required, and variouschanges, modifications, and functional additions can be thus madewithout departing from the scope of the present invention.

INDUSTRIAL APPLICABILITY

the present invention, a detection of an analyte that is provided with awide dynamic range can be carried out in a precise manner in a field inwhich a detection of a higher degree of accuracy is required such as aclinical trial of a blood test or the like using a surface-plasmonenhanced fluorescence spectroscopy (SPFS) for instance.

REFERENCE SIGNS LIST

-   10: SPFS apparatus-   12: Dielectric member-   12 a: Upper surface-   12 b: Side surface-   14: Metallic thin film-   14 a: Upper surface-   16: Fine flow passage-   18: Sensor chip-   20: Sensor chip mounting part-   22: Sensor part-   24: Light source-   26: Excitation light-   28: Fluorescence-   30: Light detection means-   32: Fluorescence amount adjusting means-   32 a: Fluorescence amount adjusting means-   32 b: Fluorescence amount adjusting means-   34: Filter

1. A surface plasmon-field enhanced fluorescence stereoscopicmeasurement method comprising the steps of exciting a fluorescencesubstance that has labeled an analyte by surface plasmon light that hasbeen generated by applying an excitation light to a metallic thin filmand receiving the generated fluorescence by a light detection means tothereby detect the analyte, wherein: a dynamic range is expanded byadjusting a light amount of the fluorescence that is received by thelight detection means.
 2. The surface plasmon-field enhancedfluorescence stereoscopic measurement method as defined in claim 1,further comprising the steps of: by comparing a first fluorescencesignal that is output in the case in which the light detection meansreceives a fluorescence under a first condition and a secondfluorescence signal that is output in the case in which the lightdetection means receives a fluorescence in which a light amount has beenadjusted under a second condition in which a light amount of afluorescence that is received by the light detection means is adjustedso as to be smaller than that in the first condition, judging whether ornot the first fluorescence signal is abnormal; and obtaining a normalfluorescence signal by correcting the second fluorescence signal in thecase in which it is decided that the first fluorescence signal isabnormal.
 3. The surface plasmon-field enhanced fluorescencestereoscopic measurement method as defined in claim 1, furthercomprising the step of adjusting a light amount of a fluorescence thatis received by the light detection means by adjusting a light amount ofthe fluorescence that has been generated.
 4. The surface plasmon-fieldenhanced fluorescence stereoscopic measurement method as defined inclaim 1, further comprising the step of adjusting a light amount of afluorescence that is received by the light detection means by adjustinga light amount of the excitation light.
 5. The surface plasmon-fieldenhanced fluorescence stereoscopic measurement method as defined inclaim 1, further comprising the step of: receiving a fluorescence by thelight detection means while changing an incidence angle of theexcitation light to the metallic thin film in a predetermined range; andjudging an abnormal fluorescence signal based on the relationshipbetween an incidence angle of the excitation light and a fluorescencesignal that is output in the case in which the light detection meansreceives a fluorescence.
 6. A surface plasmon-field enhancedfluorescence spectroscopic measurement apparatus that is configured tocarry out a detection of a specimen material by applying an excitationlight, in which a metallic thin film that is formed on a dielectricmember, a fine flow passage that is formed on an upper surface of themetallic thin film, and a sensor chip that is provided with a sensorpart that is formed in the fine flow passage are mounted, comprising: alight source that is configured to apply an excitation light to themetallic thin film via the dielectric member; and a light detectionmeans that is disposed over the sensor chip, wherein the light detectionmeans is configured to receive a fluorescence that is generated byexciting a fluorescence substance that labels an analyte that is fixedto the sensor part by a surface plasmon light that is generated in thecase in which the excitation light is applied to the metallic thin film;and a fluorescence amount adjusting means is configured to be able toadjust a light amount of a fluorescence that is received by the lightdetection means.
 7. The surface plasmon-field enhanced fluorescencespectroscopic measurement apparatus as defined in claim 6, wherein: bycomparing a first fluorescence signal that is output in the case inwhich the light detection means receives a fluorescence under a firstcondition and a second fluorescence signal that is output in the case inwhich the light detection means receives a fluorescence in which a lightamount has been adjusted by the fluorescence amount adjusting meansunder a second condition in which a light amount of a fluorescence thatis received by the light detection means is adjusted so as to be smallerthan that in the first condition, it is judged whether or not the firstfluorescence signal is abnormal; and a normal fluorescence signal isobtained by correcting the second fluorescence signal in the case inwhich it is decided that the first fluorescence signal is abnormal. 8.The surface plasmon-field enhanced fluorescence spectroscopicmeasurement apparatus as defined in claim 6, wherein the fluorescenceamount adjusting means is disposed between the sensor chip and the lightdetection means.
 9. The surface plasmon-field enhanced fluorescencespectroscopic measurement apparatus as defined in claim 6, wherein thefluorescence amount adjusting means is disposed between the light sourceand the dielectric member.
 10. The surface plasmon-field enhancedfluorescence spectroscopic measurement apparatus as defined in claim 6,further comprising an application angle adjusting means that isconfigured to adjust an incidence angle of the excitation light to themetallic thin film.
 11. The surface plasmon-field enhanced fluorescencespectroscopic measurement apparatus as defined in claim 6, wherein: afluorescence is received by the light detection means while changing anincidence angle of the excitation light to the metallic thin film in apredetermined range by using the application angle adjusting means; andan abnormal fluorescence signal is judged based on the relationshipbetween an incidence angle of the excitation light and a fluorescencesignal that is output in the case in which the light detection meansreceives a fluorescence.